PROJECT SUMMARY Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth, with long term sequelae in survivors and no curative treatment. The molecular mechanisms that promote the BPD phenotype of impaired alveolarization and fibrosis are not known, and this knowledge gap impedes the development of new BPD therapies. BPD primarily affects preterm infants born during the vulnerable saccular stage of lung development (23-32 weeks gestation), with resulting injury from hyperoxia, inflammation, and mechanical stretch. Our prior work has focused on understanding how saccular stage injury disrupts the normal patterning of developmental pathways. One of these signaling pathways, Wnt, has peak activation in the canalicular stage and gradually decreases during the saccular stage, with little active Wnt signaling present at term birth. We recently demonstrated aberrant activated Wnt signaling in the lungs of infants with BPD. Moreover, exogenous activation of Wnt signaling in our 3D human model and our hyperoxia mouse model is sufficient to reproduce the BPD phenotype in vitro and ex vivo, suggesting that Wnt is an important driver of BPD pathogenesis. Transforming growth factor-??(TGF-?) is another developmental pathway necessary for normal lung development that in excess contributes to impaired alveolarization and fibrosis. Exposure of saccular stage lung to hyperoxia injury results in increased activated Wnt and TGF-? signaling, and we are able to experimentally induce TGF-? signaling by activation of Wnt alone. Our preliminary data show that hyperoxia exposure of saccular stage lung results in increased mesenchymal expression of ligand Wnt5A as well as increased epithelial expression of Wnt modulator r-spondin-3 (RSPO3), suggesting spatial specificity of Wnt ligand expression in this model. We therefore hypothesize that that injury during the saccular stage of lung development causes aberrant epithelial expression of RSPO3 and aberrant mesenchymal expression of Wnt5A, and this increased Wnt signaling facilitates the activation of downstream TGF-? signaling, resulting in impaired alveolarization and fibrosis seen in BPD. Our specific aims are to: 1) determine the role of Wnt signaling in mediating impaired alveolarization and fibrosis in BPD, and 2) to identify the mechanisms whereby hyperoxia exposure results in Wnt pathway activation and up-regulation of TGF-? signaling. Successful completion of these specific aims will improve our understanding of the contribution of Wnt signaling to the injury response in the developing lung and provide a foundation for targeted therapies to prevent BPD. Another major objective of this proposal is to create a focused and structured career development plan that will provide the necessary training for the researcher to become an independent physician-scientist with expertise in the molecular mechanisms of BPD. The experiments and training program in this proposal create the structure for the growth of an independent rigorous research program focused on developmental lung diseases.